Image-enabled cell sorting permits prospective isolation of primitive hematopoietic stem cell subsets with distinct...

Fluorescence-activated cell sorting (FACS) has been a core technology for the identification, characterization and isolation of hematopoietic stem cells (HSCs), allowing for prospective isolation of cells which express specific molecules at specific levels of intensity. A recent technological breakthrough, image-enabled cell sorting (ICS), is based on FACS but it also permits separation of cells based on the differential distribution of these molecules across the cell. Using ICS in primary HSCs, we surveyed the patterns of expression for a range of intracellular markers and identified mitochondrial dyes as a tool to prospectively isolate HSCs with different properties. Integration of index-sort data with single-cell divisional kinetics identified total intensity and diffusivity of the MitoTracker dye as parameters enabling prospective isolation of functionally heterogeneous HSCs. In vivo functional assays confirmed that HSCs capable of long-term multilineage contribution were highly enriched in the MitoTracker low diffusivity, low intensity populations. Moreover, HSCs displaying high MitoTracker intensity but low MitoTracker diffusivity (Mito^±), displayed the characteristics of low MitoTracker intensity (Mito^-/-), representing a subpopulation of HSCs only isolatable by tools such as ICS. Collectively these data demonstrate that ICS is a powerful tool for prospectively enriching highly specific cell populations in the hematopoietic system, making it broadly useful for applications where population identification and purity are required to attain robust cellular and molecular characterization.